The biocontrol effect of Clonostachys rosea (strains 016 and 1457) on Fusarium graminearum, F. avenaceum, F. verticillioides, F. langsethiae, F. poae, F. sporotrichioides, F. culmorum and Microdochium nivale was evaluated on naturally infected wheat stalks exposed to field conditions for 180 days. Experiments were conducted at two locations in Argentina, Marcos Juarez and Río Cuarto. Antagonists were applied as conidial suspensions at two inoculum levels. Pathogens were quantified by TaqMan real-time qPCR. During the first year at Marcos Juarez, biocontrol was observed in one antagonist treatment for F. graminearum after 90 days (73% reduction) but after 180 days, the pathogen decreased to undetectable levels. During the second year, biocontrol was observed in three antagonist treatments for F. graminearum and F. avenaceum (68·3% and 98·9% DNA reduction, respectively, after 90 days). Fusarium verticillioides was not controlled at Marcos Juarez. At Río Cuarto, biocontrol effects were observed in several treatments at different intervals, with a mean DNA reduction of 88·7% for F. graminearum and F. avenaceum, and 100% reduction for F. verticillioides in two treatments after 180 days. Populations of F. avenaceum and F. verticillioides were stable; meanwhile, F. graminearum population levels varied during the first 90 days, and low levels were observed after 180 days. The other pathogens were not detected. The study showed that wheat stalks were important reservoirs for F. avenaceum and F. verticillioides populations but less favourable for F. graminearum survival. Clonostachys rosea (strain 1457) showed potential to reduce the Fusarium spp. on wheat stalks.
Fusarium head blight (FHB) is an important fungal disease of small grain cereals such as wheat and barley, causing yield losses and mycotoxin contamination of grains. The principal causal agent of FHB is Fusarium graminearum (teleomorph Gibberella zeae; lineage 7 within the F. graminearum complex; O'Donnell et al., 2004; Starkey et al., 2007), which potentially produces trichothecenes deoxynivalenol (DON) and nivalenol (NIV) (Desjardins, 2006). Other related species such as F. culmorum, F. poae, F. proliferatum, F. avenaceum and Microdochium nivale can also cause FHB (McMullen et al., 1997; Champeil et al., 2004; Osborne & Stein, 2007). In Argentina, FHB epidemics have occurred in wheat in 17 of the last 50 years, resulting in yield and quality losses (Reynoso et al., 2011).
Fusarium head blight of wheat can be controlled by chemical control or crop rotation strategies (Homdork et al., 2000; Lori et al., 2009; Mesterházy et al., 2011). Biological control has been demonstrated as an alternative strategy both in greenhouse trials and under field conditions (Jochum et al., 2006; Schisler et al., 2006; Khan & Doohan, 2009; Palazzini et al., 2009). Since the 1980s, conservative tillage practices, including no tillage, are increasingly followed in Argentina, so that crop stubble and straw residues are left on field soils. Fusarium graminearum and other Fusarium species including F. verticillioides, F. avenaceum and F. poae have a saprophytic stage and can survive in residues of crops such as maize, soyabean and wheat (Leslie & Summerell, 2006). Such colonized residues can be the inoculum sources within and outside wheat fields. In many studies, residues of previously infected crops have been found to be the main sources of spores causing head blight of wheat and maize (Dill-Macky & Jones, 2000; Shaner, 2003; Vogelgsang et al., 2011). Antagonistic microorganisms applied to crop stubbles can reduce the survival and multiplication of pathogens present in the residues and thus prevent or delay disease epidemics. Reduction of pathogen populations through biocontrol applied to crop stubble has been evaluated by Luongo et al. (2005) by using strains 016 and 1457 of the fungus Clonostachys rosea (teleomorph Bionectria ochroleuca; Schroers et al., 1999). In this study, the authors demonstrated biocontrol against various Fusarium spp. on wheat and maize stubble under controlled conditions and on stalks and maize ears under field conditions by traditional methodologies.
The objectives of the present work were to study the effects of two C. rosea strains on the populations of main pathogenic Fusarium spp. and M. nivale on wheat stalks and to evaluate the population dynamics of the pathogens on stalks. Three field experiments with naturally infected wheat stalks exposed on field soils were carried out in Argentina over two consecutive years, and the colonization of wheat pathogens was followed by using species-specific TaqMan real-time quantitative PCR (qPCR).
Materials and methods
Clonostachys rosea strains 016 (from Plant Research International (PRI), the Netherlands) and 1457 (from Istituto Sperimentale per la Patologia Vegetale (ISPaVe), Italy) were selected, based on the effectiveness of these strains in previous studies (Luongo et al., 2005). Conidia were produced on oatmeal agar (25°C for 14 days and 12 h dark/light cycle). Conidial suspensions were obtained by flooding the cultures with sterile distilled water containing Tween 80 (0·05%). Concentrations of conidial suspensions were determined by using a haemocytometer chamber and were adjusted to 106 and 104 conidia mL−1.
Microdochium nivale and Fusarium strains
Fusarium graminearum 88/790, F. poae 93-1780, F. langsethiae PRI 07-01, F. culmorum PD 90-283, F. avenaceum IPO 92-3, M. nivale 766, F. verticillioides ITEM 2142 and F. sporotrichioides ITEM 3596 were obtained from the collection of PRI. Fusarium graminearum KRC6, F. avenaceum T187 and F. verticillioides RC2096 were isolated from cereal grains from Argentina (Table 1).
Table 1. Fusarium species and Microdochium nivale used in the experiments, host origin and source
F. avenaceum IPO 92-3
F. avenaceum T187
F. culmorum PD 90-283
F. graminearum 88/790
F. graminearum KRC6
F. langsethiae PRI 07-01
F. poae 93-1780
F. sporotrichioides ITEM 3596
F. verticillioides ITEM 2142
F. verticillioides RC2096
M. nivale 766
Biomass production and DNA extraction
Pathogen species were grown in complete medium (Correll et al., 1987) and incubated on an orbital shaker (150 rpm) for 3–5 days at 25°C. The resulting mycelia were harvested by filtration through non-gauze milk filters (Ken AG). Excess water was removed by blotting mycelia between clean paper towels and dried mycelia were stored at −20°C. DNA was extracted with a cetyltrimethylammonium bromide (CTAB) method (Leslie & Summerell, 2006). Fungal DNA concentrations were determined with a spectrophotometer (NanoDrop 2000, Thermo Scientific).
Stalks were collected from commercial wheat fields located in Marcos Juarez for the 2010 and 2011 experiments and in Río Cuarto for the 2011 experiment; both areas were located in Córdoba province, Argentina. Stalk pieces 10–12 cm long containing the crown node and first node were used in the study. Commercial wheat was planted in June and harvested in early December, when stalks were collected. Wheat cultivars ProInta Halcon and Klein Escorpión were used at Marcos Juarez and Río Cuarto, respectively. Field rotation at Marcos Juarez was wheat over wheat, and at Río Cuarto the previous crop was soyabean.
Treatments and antagonist applications
Stalks were put in polyethylene net bags (20 × 20 cm; 4 mm2 mesh), each bag containing 20 stalks in the 2010 experiment and 50 stalks in the 2011 experiments. Each bag with stalks was treated as a single sample unit. Bags containing wheat stalks were submerged for 1 min in suspensions containing conidia of C. rosea or sterile distilled water. A total of five treatments were used: two strains of C. rosea (016 and 1457) at two inoculum levels (106 and 104 conidia mL−1) and a control treatment of sterile water. Treated bags were fixed on fallowed field soil with iron nets to prevent disturbance, e.g. by birds. A completely randomized block design with five blocks (replicates), five plots (treatments), each treatment consisting of two bags allowing for two sampling dates, was used in each experiment. Additionally, five bags with water-treated stalks as (controls) were stored at −18°C without exposure to field soil.
Daily mean temperature, mean humidity and total precipitation were obtained from weather stations located in the area of the field trials (INTA Marcos Juarez and Río Cuarto University).
Bags with wheat stalks were collected 90 and 180 days after being placed in the field (five control samples and 20 treated samples per collecting date). At Río Cuarto, additional sets of bags were sampled at 15, 30 and 60 days. The Río Cuarto experiment started on 20 January 2010 with sampling dates for 15, 30, 60, 90 and 180 days on 8 and 22 February, 17 March, 19 April and 20 July 2011, respectively. Sampling dates corresponding to 15, 30 and 60 days were evaluated to monitor population dynamics of the Fusarium pathogens on wheat stubble without biocontrol treatments. The Marcos Juarez 2010 experiment started on 18 December 2009 and the collecting dates for 90 and 180 days were 16 March and 12 June 2010, respectively. The Marcos Juarez 2011 experiment started on 21 January 2010 and the sampling dates for 90 and 180 days were 22 April and 16 July 2011, respectively.
Sample analysis and qPCR conditions
Each sample (bag), consisting of the residues of 20 stalk pieces, was split. One subsample was dried at 105°C for 24 h to determine the dry matter content and assess stalk decomposition (dry weight loss). The other subsample was freeze-dried and pulverized in a mill with a 1 mm2 mesh screen (Cyclotech, Foss Tecator). DNA was extracted from approximately 10 mg of the pulverized subsamples using the DNeasy 96 plant kit (QIAGEN) with small modifications: incubation (step 8 in the manufacturer's protocol) was 30 min instead of 10 min and the DNA elution step was done twice with 50 μL instead of once with 100 μL. qPCR quantifications were done on an ABI Prism 7500 Sequence Detection System (Applied Biosystems). Separate TaqMan reactions were performed in 25 μL, using 12·5 μL of TaqMan 2× universal PCR master mix (Applied Biosystems), 100 nm of FAM-labelled probe and internal control probe, and 400 nm of forward and reverse primer for both the target Fusarium species and M. nivale (Waalwijk et al., 2004, 2008) as well as the internal positive control (Klerks et al., 2004). qPCR reactions were performed on 2 μL of DNA preparations from stalk samples and pure pathogen DNA. Thermal cycling conditions consisted of 10 min at 95°C, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Standards curves were generated by using tenfold serial dilutions of pure DNA in the range of 0·1 to 1 × 104 pg μL−1, and five replicates of pathogen quantifications were done. When analysing samples, samples of the corresponding pathogen DNA were run in parallel using a tenfold serial dilution ranging from 0·1 to 1 × 104 pg μL−1 as reference. From the obtained Ct values of qPCR for the different pathogen DNA dilution series and for DNA extracts from wheat stalk samples, the concentrations of pathogen DNA in the samples were calculated, expressed as pg of DNA of fungal pathogen per mg of plant tissue (dry weight).
To determine the reproducibility of the assays, independent experiments were performed on different days with a selected set of samples consisting of negative samples (n =18), low pathogen DNA content samples (<100 pg DNA mg−1 dry wheat stalk, n =15) or high pathogen DNA content samples (>100 pg DNA mg−1, n =10) of F. avenaceum and F. verticillioides. Robustness of the qPCR quantifications was analysed at two levels. First, quantifications were performed on two independent DNA extractions of this set of samples; and secondly, quantifications of samples were done at three different positions in the 96-well plate. This resulted in a coefficient of variation (CV, in percentage) of the original scale.
Experiments were analysed separately. Data were transformed to natural logarithm before analysis of variance (anova). Means of DNA concentrations of the various Fusarium spp. for the different treatments and sampling dates were separated with Holm–Sidak method (P ≤ 0·001). All statistical analyses were done using sigmastat for windows v. 3.5 (SPSS Inc.).
Validation of qPCR measurements
In the cases where the quantification of the internal control was underestimated indicating PCR inhibition, qPCR runs were repeated with diluted DNA suspensions. New qPCR runs with diluted DNA samples (maximum dilution was 1/20) resulted in no inhibitions of the internal control and Ct values for the internal control were always similar (Ct = 30·4 ± 0·7). Quantifications of DNA extracted independently from the selected set of samples (n =43) gave an overall CV of 20·41% on the original scale. For samples with values >100 pg DNA mg−1 (n =10), quantified on different days, an averaged CV of 3·66% was observed. For samples with values < 100 pg DNA mg−1 (n =15), the CV was higher (35·32%). When analysing quantifications of the same sample in different well positions on the same run, averaged CV values were 8·86% for samples with high DNA content (n =10) and 47·97% for samples with low DNA content (n =15). The absence of pathogen DNA (negative samples, n =18) was always confirmed by the measurement of a second subsample.
Biocontrol effect at Marcos Juarez
At the beginning of the 2010 field trial, high amounts of F. graminearum, F. avenaceum and F. verticillioides DNA were quantified, with mean levels of 424, 224 and 1702 pg DNA mg−1, respectively (backtransformed values). Fusarium poae was also detected, but below the level for reliable quantification (quantification limit 0·1 pg DNA mg−1). No amplification of F. culmorum, F. sporotrichioides, F. langsethiae or M. nivale DNA was detected. After 90 days, F. graminearum, F. verticillioides and F. avenaceum DNA levels had decreased significantly and remained low until the end of the experiment (180 days; Fig. 1a). Other pathogens detected at the beginning of the experiment remained undetectable after 90 days and were not analysed after 180 days. A biocontrol effect was only observed against F. graminearum when C. rosea strain 1457 was applied at 1 × 106 spores mL−1 (Cr1457–106), significantly reducing the concentration of F. graminearum DNA from 4·88 (control treatment) to 1·32 pg DNA mg−1 (73% reduction) after 90 days (Fig. 2). After 180 days, F. avenaceum and F. verticillioides were present in control treatments with 5·2 and 52·4 pg DNA (backtransformed values) detected, respectively. No biocontrol effect was observed and no differences were observed either between treatments and controls or between controls at 90 or 180 days for F. avenaceum or F. verticillioides (Fig. 2b,c). Temperatures were moderate during the first half of the experiment (mean temperature from December 2009 to March 2010 was 23·4°C) and 14·3°C during the second half (April–June 2010). Precipitation during the experiment was low (61·2 mm).
In the Marcos Juarez 2011 experiment, lower amounts of F. graminearum, F. avenaceum and F. verticillioides DNA were quantified at the beginning in comparison with the 2010 field trial (Fig. 1b). Fusarium poae was detected in two of five control plots, with a mean DNA level of 108·5 pg at the beginning of the experiment, but after 90 days the mean DNA level was 25 pg and only detected in one of five control plots. No amplification of F. culmorum, F. sporotrichioides, F. langsethiae or M. nivale DNA was detected. A similar pattern of decline in DNA was observed for F. avenaceum and F. verticillioides in 2011 as in 2010. No statistical differences were observed for F. avenaceum DNA quantifications between the initial and 90 days samplings. A small but not statistically significant decrease was observed in F. avenaceum between 90 and 180 days (Fig. 1b). After 90 days, F. graminearum decreased to low DNA levels and was not detected after 180 days. Treatments Cr016–106, Cr1457–106 and Cr1457–104 statistically reduced F. graminearum DNA measured after 90 days (68·3% overall reduction) but it was not possible to evaluate at 180 days because the pathogen DNA had decreased to undetectable levels (Fig. 3a). In contrast to F. graminearum, F. verticillioides and F. avenaceum control treatments maintained high DNA levels until 180 days (18·22 and 11·74 pg DNA mg−1, respectively). A biocontrol effect was observed for F. avenaceum in treatments Cr016–106, Cr1457–106 and Cr1457–104 after 90 days (98·9% overall reduction) and in Cr016–106 and Cr1457–104 after 180 days (96·4% overall reduction; Fig. 3b). No biocontrol effect was observed against F. verticillioides at Marcos Juarez during the 2 years of experiments (Figs 2c and 3c). Temperatures during the experiment were similar to 2010 (23·2°C from January to March and 16·8°C from April to June) but total precipitation was 357 mm.
Additionally, stalk decomposition was analysed based on dry weight loss during the experiments but no differences were observed between treatments and controls after 90 or 180 days (data not shown).
Biocontrol effect and population dynamics of Fusarium species at Río Cuarto
Initial quantities of F. graminearum, F. avenaceum and F. verticillioides DNA were similar, ranging from 46 to 72 pg DNA mg−1 (control treatments; Fig. 1c). Fusarium poae was detected in one of five control plots, with a DNA level of 74 pg at the beginning of the experiment, and was not detected after 90 days. No amplification of F. culmorum, F. sporotrichioides, F. langsethiae or M. nivale DNA was detected at the beginning of the experiment. After 90 days, F. graminearum DNA decreased to 2·4 pg mg−1 in the control and a significant biocontrol effect was observed in Cr016–106 and Cr1457–104 treated plots (85·9% overall reduction). After 180 days, DNA values for F. graminearum in treatments and control were close to the quantification limit (0·1 pg DNA mg−1). A biocontrol effect was also observed for F. avenaceum in treatments Cr016–106 and Cr1457–104, with DNA reductions from 91·6 to 100% after 90 or 180 days. Fusarium verticillioides was reduced by treatments Cr016–106 and Cr1457–104 after 180 days, when no pathogen DNA quantification was obtained (Fig. 4).
Populations of F. verticillioides and F. avenaceum increased during the first 60 days from 47·4 to 206·4 pg DNA mg−1 (without significant differences between different sampling dates) and remained stable during the 180 day period of the experiment. However, the F. graminearum population decreased significantly between the initiation of the experiment and the 15 day sampling, and between the 60 and 90 day samplings. Fusarium graminearum DNA was close to zero at the end of the experiment (Fig. 1c).
Temperatures during the experiment were similar to the Marcos Juarez experiments during the first half of the trial (mean January to March temperature of 19·8°C) and slightly lower during the second part of the experiment (10·9°C from March to June). Total precipitation was 428 mm.
The biocontrol effect of C. rosea strains 016 and 1457 against Fusarium spp. pathogens was studied on wheat stalks over 2 years at Marcos Juarez and 1 year at Río Cuarto, Córdoba province, Argentina. Additionally, the population dynamics of three Fusarium pathogens was analysed at Río Cuarto over a period of 180 days. In general, higher concentrations of both biocontrol agents were more effective in controlling pathogen populations than lower concentrations. A better biocontrol effect of C. rosea strain 1457 was also observed. The effectiveness of C. rosea is attributed to its ability to colonize senescent and dead tissue faster than the pathogen and to suppress pathogen sporulation (Morandi et al., 2000, 2001) and, in this study, it is possible that the C. rosea strains used could prevent the recolonization of wheat stalks by the Fusarium pathogens evaluated. Biocontrol activity of C. rosea against several pathogens, mainly Botrytis cinerea in rose, rose debris and other crops such as strawberry, carrots and wheat seeds have been reported (Tatagiba et al., 1998; Morandi et al., 2000; Nobre et al., 2005; Cota et al., 2008; Roberti et al., 2008). Isolate C. rosea strain 016 used in the present study was demonstrated to control B. cinerea in cyclamen under commercial growing conditions in an earlier study (cited as Gliocladium roseum 1813 by Köhl et al., 1998). Clonostachys strains used in the present study have been previously evaluated by Luongo et al. (2005) against several Fusarium pathogens in experiments under controlled conditions with wheat and maize stalks inoculated artificially after irradiation and in field assays on maize stalks and ears. These strains were effective in reducing pathogen sporulation by 60–90% on stalks in controlled environment experiments. In field experiments on maize stalks pieces, C. rosea strain 1457 was effective in diminishing F. verticillioides and F. proliferatum in different years and F. graminearum in both years on maize grains. However, a direct comparison of the results obtained in the two studies is not possible because different parameters, e.g. spore counts versus total DNA concentrations, have been measured to assess biocontrol effects.
At Marcos Juarez, high levels of F. graminearum, F. avenaceum and F. verticillioides DNA were quantified at the beginning of the 2010 experiment, which can be explained by the favourable climatic conditions prior to the sampling, with a mean temperature from October to December 2009 of 21·5°C and an accumulated precipitation of 247 mm. During the experiment, a significant biocontrol effect of C. rosea strain 1457 was observed against F. graminearum at an inoculum level of 106 but no effect was detected against F. verticillioides and F. avenaceum populations 180 days after placement in the field.
In the Río Cuarto experiment, the most effective treatments were Cr016–106 and Cr1457–104. However, Cr1457–106 (high spore level) was not effective for unknown reasons. Fusarium graminearum controls showed the same behaviour observed at Marcos Juarez, and decreased from 72·8 pg DNA mg−1 to low levels after 90 days (<2·4 pg DNA mg−1) and 0·27 pg DNA mg−1 after 180 days (near quantification limit). Fusarium verticillioides remained at high DNA level whereas F. avenaceum decreased after 180 days (23·85 pg DNA mg−1, 50% of the initial level).
Populations of F. avenaceum and F. verticillioides were relatively stable in the Río Cuarto experiment, but F. graminearum declined to very low levels within 90 days. During the first 30 days, an accumulated precipitation of 230 mm and a mean temperature of 22·6°C were recorded. These climatic conditions seemed favourable for F. avenaceum and F. verticillioides maintenance, but not for F. graminearum. It is known that F. graminearum is not a strong saprophytic colonizer and can be replaced by other more effective decomposers, especially in wheat tissues (Bowen, 1990). The dynamics of F. graminearum in residues of wheat exposed to the surface or buried was also observed by Pereyra et al. (2004) during a 2-year experiment. After the first year, the authors found 80% survival in nodes exposed to the surface and approximately 60% in buried residues; they concluded that ascospores can be produced in wheat residue and remain active to contribute to new infections for the next crop. In the Marcos Juarez 2010 experiment, F. graminearum inoculum (measured as DNA) disappeared after 180 days, but it was present in stubble for the following crop at high levels. It is therefore possible that sufficient amounts of inoculum remained viable until the next year, or wind transported spores arrived at the field, which is strongly supported by several studies (Maldonado-Ramirez et al., 2005; Schmale et al., 2005; Schmale, 2006). The latter option is also valid for the prevalence of F. graminearum inoculum observed in previous studies (Pereyra et al., 2004; Schmale, 2006). The dynamics of the FHB pathogens and M. nivale was studied by Köhl et al. (2007) in crops and crop residues in winter wheat assessed by TaqMan qPCR. The authors found a significant decrease of the pathogens in internodes and nodes of wheat residues after 10 months, but not in stem base residues. This correlates with the results observed for F. avenaceum and F. verticillioides in the current study. The authors suggested that the stem base can act as a residue for the long term survival of inoculum of Fusarium spp. Wheat stalk segments used in the present study contained the crown node and the first node, as previously analysed by Köhl et al. (2007). Pereyra & Dill-Macky (2008) analysed the presence of Gibberella zeae in artificially inoculated wheat, maize, barley and sunflower grown in minimum or no tillage systems. They found a high incidence of the pathogen at harvest time in the crops but reported a decline in the crop residues during the following 2 years. Wheat and barley residues produced more ascospores in comparison to maize. Similarly, Lori et al. (2009) analysed the occurrence of FHB in wheat grown in different tillage systems under natural infection over 3 years. Climate conditions had a stronger effect on FHB than tillage conditions. During the Marcos Juarez 2010 experiment, mean temperatures were moderate and precipitation was extremely low. It is possible that the prevailing climatic conditions during the field experiment did not support Fusarium spp. development. Fusarium verticillioides is an important maize pathogen (Pereira et al., 2007), able to produce fumonisins that cause several diseases in animals and are associated with oesophageal and liver cancer in humans (Stankovic et al., 2012). This pathogen was present in wheat stalks in high amounts. As wheat is commonly grown in Argentinean fields in rotation with maize or soyabean (Lori et al., 2009) using minimum or no tillage practices (Edwards, 2004; Lori et al., 2009), wheat stubble can maintain the inoculum of F. verticillioides and increase the risk for other crops grown later in rotation in the same field. As maize, wheat and soyabean are the most important grain crops in Argentina (Pereira et al., 2007; Reynoso et al., 2011), special attention must be paid to diminish the pathogen inoculum. Adequate rotation of these crops associated with biocontrol or other management strategies could reduce the risk of crop infection and mycotoxin accumulation in commercial commodities.
This is the first report from Argentina on biocontrol and Fusarium species dynamics on wheat stubbles assessed by qPCR. The results showed strong effectiveness (68–100% pathogen DNA reduction) of a single application of the biocontrol agents assayed up to 6 months later. In control treatments, the F. graminearum populations naturally decreased and F. avenaceum and F. verticillioides populations were relatively stable over 6 months exposure in two fields and in both years under simulated no tillage conditions. Further experiments are needed to evaluate the effectiveness of C. rosea isolates in different fields under different climatic conditions on wheat stalks and on the development of diseases caused by Fusarium spp. in the subsequent crops.
This work was supported by EC KBBE-2007-222690-2 MYCORED.